Chemiluminescent measurement of peroxidase activity and its application using a lucigenin CT-complex

Luminol (3-aminophthaloylhydrazine) is well known as a chemiluminescent rea gent that can be used to measure peroxidase (POD) activity. We have develop ed a more sensitive assay for POD using a new chemiluminescent reagent base d on 10,10'-dimethyl-9,9'-biacridinium nitrate (lucigenin). This new reagen t was obtained from lucigenin by light irradiation in an organic polar solv ent such as N,N-dimethylacetamide (DMAc). The detection limit for POD activ ity using this reagent is 10(-19) mol/assay. In an application for enzyme i mmunoassay based on a POD label, we were able to detect human alpha-fetopro tein (AFP) at 1 pg/mL. The characterization of this chemiluminescent reagen t was obtained by optical measurements, and the formation of lucigenin char ge transfer complex (CT-complex) was confirmed. We also investigated the st ability of lucigenin CT-complex as a chemiluminescent reagent for the measu rement of POD activity, and confirmed that the reagent was stable for more than a year. Copyright (C) 2000 John Wiley & Sons, Ltd.